There has been a long-standing need for development of serum-free culture media for various types of mammalian cells in vitro. Inherent in this development is the ability to stably move exogenous genetic information into various cell types to study the effects of homologous and non-homologous genes by the construction of stable genetic recombinant cell lines. Advantages of serum-free culture systems for the study of human somatic cell genetics include the following: (1) access to epithelial cells without the complicating factor of feeder-cells and fibroblasts; (2) the ability to study gene expression after transfection with genes that are controlled by host factors and hormones in the culture medium; (3) a reduction in experimental variation caused by the performance of serum in cell growth and gene expression; and (4) the economic benefit obtained by producing biologically active products from selected cell constructs without the need to introduce a contaminant requiring removal before the product can be used.
The polyethylene glycol (PEG) fusion method routinely used for production of mammalian cell hybrids and for transient cell expression experiments in mammalian and HeLa cells is most difficult to use as described for stable genetic transfection of human cells. See particularly Schaffner, Sandri-Goldin et al, and Rassoulzadegan et al in the Bibliography for examples of the PEG fusion method. Among other changes, this improved protocol requires the development of a new PEG fusion reagent--the subject of this invention. Removal of the toxic components of the PEG by treatment with ion exchange resins results in a reagent that is non-toxic when applied as a fusogen to normal human cells in culture. The procedure followed, and the PEG reagent used, successfully yields efficient genetic transfection of the following types of human cells: epithelial cells, mesynchemial cells, fibroblast cells, and hematopoetic cells. The types of genes used in these experiments include human virus genes linked to pSV2gpt, proviral and cellular oncogenes carried on pBR322, and human DNA libraries linked to pSV2neo.
Genetic analyses of mammalian cells have utilized cell-fusion methods to construct genetic hybrids for a number of types of tests requiring the formation of interspecies cell-cell hybrids [Pontecorvo, G; Somatic Cell Genetics, Vol. 1, p 397-400 (1975)], and transfer of genes carried on bacterial plasmids to mammalian cell recipients. The application of fusion technology to problems relating to human somatic cell genetics has proven difficult since many human cell types are sensitive to the cytotoxic contaminants in one of the most commonly used fusion reagents, polyethylene glycol (see Bibliography). Methods to circumvent or reduce the effect of the toxic components of polyethylene glycol (PEG) include the use of longer polymeric chain length PEG (3000 to 6000 MW) as a replacement for the more efficient membrane fusogen PEG-1000, shortened treatment times, and screening PEG lots for cytotoxic effects before selecting the least toxic reagent for application to cell fusion procedures. The present invention discloses an ion exchange resin treatment of polyethylene glycol-1000 which renders the reagent virtually non-toxic when applied as described here at 48% weight/weight (w/w) concentration to normal human fibroblast cells grown in culture. This improved fusogen provides an efficient reagent for application to human cells without causing notable cytotoxicity. This permits efficient application of fusion procedures to human cells in culture that were previously difficult or impossible to treat in vitro, and provides a reagent that will be generally useful to human somatic cell genetic analyses that involve cell membrane fusion.